Multichannel microscale system for high throughput preparative separation with comprehensive collection and analysis

ABSTRACT

While the present invention has been described in conjunction with a preferred embodiment, one of ordinary skill, after reading the foregoing specification, will be able to effect various changes, substitutions of equivalents, and other alterations to the compositions and methods set forth herein. It is therefore intended that the protection granted by Letters Patent hereon be limited only by the definitions contained in the appended claims and equivalents thereof.

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application is a divisional of U.S. application Ser. No.09/530,118 filed Apr. 24, 2000, entitled A MULTICHANNEL MICROSCALESYSTEM FOR HIGH THROUGHPUT PREPARATIVE SEPARATION WITH COMPREHENSIVECOLLECTION AND ANALYSIS which claims priority from U.S. ProvisionalPatent Application No. 60/062787, filed Oct. 24, 1997, the whole ofwhich are hereby incorporated by reference herein.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0002] Part of the work leading to this invention was carried out withUnited States Government support provided under a grant from theDepartment of Energy, Grant No. DE-FG02-90ER60985. Therefore, the U.S.Government has certain rights in this invention.

BACKGROUND OF THE INVENTION

[0003] Following the use of most modern separation techniques, furthertreatment of the separated components of a sample is required to obtainmore complete information about the nature of the components. Forexample, methods of functional genomics (e.g., differential display(Liang et al., Science 257:967-971, 1992), AFLP (Vos et al., Nucl. AcidRes. 23:4407-4414, 1995), etc.) produce a pattern of separated DNAfragments, but the products of differentially expressed genes have to beidentified separately. As another example, methods to discriminatemutations such as constant denaturant capillary electrophoresis (CDCE)also require subsequent determination of the specific mutation (Khrapkoet al., Nucl. Acid Res. 22:364-369, 1994). To perform such amultidimensional analysis, a high throughput preparative separationsystem capable of collecting comprehensively all components of thesample mixture would be desirable.

[0004] Current micropreparative techniques for purification and fractioncollection generally use either chromatography or electrophoresis forseparation of the sample components. Fully automated single columnsystems are available, allowing fractionation and collection of specificsample components per run (Karger et al., U.S. Pat. No. 5,571,398(1996); Carson et al., U.S. Pat. No. 5,126,025 (1992)). When fractionsfrom multiple lanes are required, e.g., of DNA fragments, slab gelelectrophoresis can be used for the simultaneous separation of thesamples, followed by manual recovery of the desired fractions from thegel. This process is slow, labor intensive and imprecise. In anotheranalytical approach, DNA fragments can be collected onto a membraneusing direct transfer electrophoresis (Richterich et al., Meth. Enzymol.218:187-222 1993). However, recovery of the samples from the membrane isslow and difficult.

BRIEF SUMMARY OF THE INVENTION

[0005] The invention is directed to a modular multiple lane or capillaryelectrophoresis (chromatography) system that permits automated parallelseparation and comprehensive collection of all fractions from samples inall lanes or columns, with the option of further on-line automatedsample analysis of sample fractions. At its most basic, the systemincludes a separation unit such as a capillary column having each endimmersed in a buffer solution, the inlet end being immersed in a regularbuffer tank and the outlet end being in connection with the appropriatemulti-well collection device. The outlet end may also be connected to asheath flow generator. The capillary column, which may or may not havean inner coating and may be open tube or filled with any of a variety ofdifferent separation matrices, is used for separation of mixtures ofcompounds using any desired separation technique. The term “capillarycolumn” is meant to include a vessel of any shape in which amicroseparation technique can be carried out. For example, other typesof separation units, such as channels in a microchip or othermicrofabricated device, are also contemplated.

[0006] Depending on the separation method chosen, a sample mixture couldbe introduced into one or more separation lanes simultaneously, using anelectric field, or pressure, vacuum, or gravitational forces. Fractionsusually are collected regardless of the sample composition in fixed timeintervals, preferably every few seconds, into, e.g., a multi-well platewith fixed well volume, preferably, e.g., 0.5-10 microliter or smaller.The multi-well plate has sufficient capacity to collect all possiblefractions during a separation run. Determination of sample separationprofile(s) is accomplished by monitoring, e.g., an opticalcharacteristic of the sample components, for example, laser inducedfluorescence, color, light absorption (UV, visible or IR), usingon-column or on-lane detection. After the run is completed, the desiredfractions are selected using sample profiles recorded during theseparation experiment. Determination of sample separation profile andselection of fractions may also be achieved in a post-process procedure,where collected fractions are scanned in a separate optical devicecapable of registering a desired optical characteristic of the collectedmaterial. Fractions of interest are transferred to microtubes orstandard microtiter plates for further treatment.

[0007] The multi-well fraction collection unit, or plate, is preferablymade of a solvent permeable gel, most preferably a hydrophilic,polymeric gel such as agarose or cross-linked polyacrylamide. Apolymeric gel generally useful in the system of the invention is anentangled or cross-linked polymeric network interpenetrated by asuitable solvent so that the final composition has the requiredphysico-chemical properties, e.g., sufficient electric conductivity(for, e.g., CE systems), rigidity and dimensional and chemicalstability, to serve as the collection unit of the system of theinvention. The polymer may or may not be cross-linked and may be linearor branched. Examples of suitable materials include, e.g., agarose,polyacrylamide, polyvinylpyrrolidone, polyethyleneglycol orpolyvinylalcohol, and copolymers or combinations thereof. Other suitablematerials for a collection unit include electrically conductive plasticor assemblies of micelles. The pore size(s) of gel network pores can beestablished as appropriate by modulating parameters such as polymertype, concentration, cross-linking agents and polymerization conditions.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWING

[0008] Other features and advantages of the invention will be apparentfrom the following description of the preferred embodiments thereof andfrom the claims, taken in conjunction with the accompanying drawings, inwhich:

[0009]FIG. 1a shows one embodiment of the micropreparative fractioncollection system of the invention;

[0010]FIG. 1b shows an alternative embodiment of the micropreparativefraction collection system of the invention;

[0011]FIG. 2 shows an alternative embodiment of the micropreparativefraction collection system of the invention;

[0012]FIG. 3 shows another alternative embodiment of themicropreparative fraction collection system of the invention;

[0013]FIG. 4 shows a portion of a micropreparative fraction collectionsystem according to the invention connected to a matrix replacementport;

[0014]FIG. 5 shows a portion of a micropreparative fraction collectionsystem according to the invention comprising a solvent permeable gelcollection unit in contact with a separation capillary array;

[0015]FIG. 6 shows an alternative embodiment of the micropreparativefraction collection system of the invention;

[0016] FIGS. 7A-7E show a scheme for preparing a solvent permeable gelcollection unit for use in the micropreparative fraction collectionsystem of the invention;

[0017]FIGS. 8A and 8B show two possible orientations of a separationcapillary with respect to a well of a multi-well collection unit in themicropreparative fraction collection system of the invention;

[0018]FIGS. 9A and 9B show two alternative configurations of individualwells of a multi-well collection unit in the micropreparative fractioncollection system of the invention;

[0019] FIGS. 10A-10D show a top view, side view and spacing ofindividual wells of the alternative well configuration of FIG. 9B;

[0020]FIG. 11 is a graph showing real time on-column detection of asample fractionated using the system of the invention;

[0021]FIGS. 12A and 12B are individual fraction profiles of fractionscollected from the column of FIG. 11; and

[0022]FIG. 13 shows on-column detection, individual fraction profilesand a profile of pooled fractions from the analysis of another sampleusing the system of the invention.

DETAILED DESCRIPTION OF THE INVENTION

[0023] The system of the invention will now be described in detail usingas an example a system for the separation of fluorescently labeled DNAfragments by capillary electrophoresis; however, such a system can beapplied to different sample materials, e.g., proteins, otherbiopolymers, and low molecular weight compounds, or can incorporateother separation methods, as well. Any method of separation could beemployed, including but not limited to capillary electrophoresis (CE),capillary isoelectric focusing (CIEF), capillary electrochromatography(CEC) and capillary liquid chromatography (CLC). Furthermore, anydetection parameter would be useful in the method of the invention, suchas, e.g., laser induced fluorescence, color, light absorption (UV,visible or IR), radioactivity or conductivity.

[0024] A basic micropreparative fraction collection system consists ofthe following main sections, as shown in FIG. 1a: a sample applicationunit (10), a separation unit of parallel capillary columns (20), adetection unit (30) and a multi-well fraction collection unit (40). Inthe orientation shown, i.e., with the capillaries positioned vertically,the system is most suitable for CLC analysis. For DNA fragments,separation can be performed using, e.g., fused silica capillaries filledwith a separation matrix, e.g., agarose or linear polyacrylamidesolution. As shown in FIG. 1b, separation can also be carried out inchannels (25) in microfabricated chips. Such channels preferably wouldterminate in small tips (27) for ease of transfer of individualfractions to the multi-well fraction collection unit. Referring again toFIG. 1a, the DNA samples are injected in parallel from the sampleapplication unit (10) at the cathode side of the separation unit (20),separated in the separation unit and collected at the anode side of theseparation unit into the fraction collection unit (40). The collectionperiod is determined depending on the desired resolution and speed ofseparation.

[0025] The preferred collection unit is an array of wells, each wellcapable of holding a fixed volume, e.g., ˜1 μL, that is formed in amedium such as glass, plastic, polymer or gel plate. For example, anarray plate with 5,400 wells per channel would be capable of collectingfractions for up to 1.5 hours with zone resolution of 1 sec. In general,the collection unit, preferably capable of collecting fractions havingvery small volumes, is constructed so as to maintain fraction solventevaporation at a very low level. For separation methods that use anelectric field, the fraction collection unit is in electrical contactwith the separation channels. In addition, the collection unit ispreferably biocompatible and disposable.

[0026] Two different detection systems are contemplated in the preferredsystem for identification of the desired fractions in the wells. In thefirst option, as shown in FIG. 2, an on-column detection unit (30),e.g., for laser induced fluorescence detection with a CCD camera orother image acquisition device, is positioned near the exit end of theindividual capallaries in the separation capillary array (20). Thesignal from the detector is evaluated by a computer. Based on the knowndistances between the detection point and capillary exit, and the rateof fraction deposition into the array of wells in collection unit (40),the precise position of each collected fraction of interest in themulti-well plate can be determined.

[0027] The second option, shown in FIG. 3, is based on post-collectionscanning of the array well plate. In this configuration, a laser beamfrom an LIF scanner (60) scans rows of wells across the collectionplate. Either real time scanning, in which the content of the depositedfractions is probed during the collection, or post run scanning can beused. After collection, fractions of interest can be transferred to astandard 96 or 384 well plate and amplified and sequenced using arobotic system and a DNA sequencer.

[0028] For repetitive use of the capillary array, e.g., for CE,replacement of the separation matrix may be necessary. Referring to FIG.4, the capillary array (20) may consist of two serial arrays (22, 24)aligned in a matrix replacement port (70). Replacement port (70) is ablock of, e.g., glass or plastic having an internal cavity (72), wherethe ends of the capillaries in the two serial arrays are aligned at anarrow junction (25), which is filled with separation matrix. Separationmatrix can be replaced as needed through an opening (74) in the matrixreplacement port. During matrix replacement, positive pressure isapplied to the matrix in junction (25) through the opening (74).Expelled matrix flows out from each end of all capillaries, and newmatrix is introduced. After replacement, the matrix replacement port canbe closed, the system reassembled and the next analysis commenced.

[0029] For collection of the individual zones as they exit theseparation capillaries, either a liquid sheath or electrokinetic meanscan be used for completion of the electrical circuit for thoseseparation methods requiring a circuit. In the first case, as shown inFIGS. 2 and 3, the capillaries end in a sheath flow generator (50) andthe sheath collection fluid, slowly flowing around the capillary ends,transports the material exiting the capillaries into individual wells(45) in the microtiter well plate collection unit (40). In theelectrokinetic aided mode, as shown in FIG. 5, individual capillaries inseparation capillary array (20) are in electrical contact (42) with thecollection plate (40), and zones exiting the capillaries are depositedon the collection plate by electrokinetic transport (e.g.,electrophoresis, electroosmosis). No sheath liquid is required in thismode of collection.

[0030] When very small sample volumes are being handled, there areserious issues, such as rapid solvent evaporation, for fractioncollection unit design. Therefore, the preferred multi-well collectionunit of the invention is constructed of an electrically conductivebiocompatible, solvent permeable material such as agarose orpolyacrylamide gel. Forms for collection gel plate casting can easily bemade, either by regular machining or by micromachining technologies. Thecollection plates may contain a large number of structures for samplecollection (wells, channels) and also for further samplehandling—desalting, filtration, enzyme reactions, etc. Similar gel basedplates can also be used for preseparation sample treatment, as a sampleapplication unit (10) (see FIG. 1a) or as an independent unit for sampleanalysis. In this respect, the gel plate would be similar to standardmicromachined devices, currently fabricated from glass, silicon orplastics. The advantages of the gel materials are that they are easilyhandled and molded to all types of desired configurations, they canreduce sample evaporation while also inhibiting sample diffusion orliquid leaking, and they can provide electrical conductivity, selectiveor complete ion permeability, and the possibility of creating deviceswith gradients of physico-chemical properties, e.g., gradients of porestructure or pH.

[0031] Examples of the system of the invention, including a gel basedplate for collection of zones exiting the separation capillaries, areshown in FIGS. 5 and 6. The gel plate (40) has an array of collectionwells (45). The plate is on a motorized stage (44), allowing itsmovement (46) relative to the capillary array (20). The ends of theseparation capillaries (42) are in direct contact with the surface ofthe gel so that the uninterrupted electric current can be applied forthe separation. Since the gel plate is electrically conductive, a singleelectrode (48) attached to the gel plate serves for electric connectionof all the separation capillaries. Other features of a preferred systeminclude a high voltage power source (47); a buffer reservoir (48); asolid state thermostat array (52), positioned to permit the control ofcapillary temperature during the separation process; and a laserillumination system (53), with associated line generator (54) and beamsplitter (55). The laser systen produces two point illumination for,e.g., laser induced fluorescence detection using a spectrograph/CCDdetector (56), which can have associated lens (57) and notch filters(58).

[0032] The zones exiting the capillaries are collected into themicro-wells on the gel plate; the wells may contain a collection fluid.Once collected, the fractions in the wells can be transferred out of thewells or processed directly in the wells. The evaporation of the liquidfrom the wells, which is a major problem in handling of minute samplevolumes, can be reduced or eliminated in this case since the gel itselfcontains a large excess of water. For especially small volumes, the gelplate is partially immersed in a solvent bath so that positive liquidflow into the gel and the wells of the gel will be maintained. Gelplates could be cast with microchannels, allowing consecutivemicrofluidic sample handling. These plates may also be used to performtwo dimensional electrophoresis or be utilized as a micro-storagedevice.

[0033] Beyond the use described above as a material from whichmicrotiter plates can be made, solvent permeable, e.g., hydrophilic,gels are useful in many different ways, such as for fabrication ofminiaturized devices for sample treatment, reaction and analysis.Microfabricated analytical devices are currently produced from standardsolid materials such as glass, certain metals, silicon, silicon resinsand other plastic materials. These materials are generally rigid andimpermeable to both ions and water. Electric conductivity is providedonly when metal or semiconductor materials are used. While the abovementioned materials can be used for fabrication of very small featuressuch as channels for sample delivery and separation, sample inlet andoutlet ports, unions, etc., some other desirable features such aspermeability for water or selective permeability for ionic speciesand/or a low absorptivity surface cannot easily be achieved in prior artdevices. Miniaturized devices fabricated of the solvent permeable gelmaterial of the invention can contain all the desirable features of thedevices of the prior art and in addition solve the problem of rapidevaporation of samples.

[0034] A miniaturized analogue of the microtiter well plate can easilybe produced by gel casting into any desired shape. Referring to FIGS.7A-7E, by using the procedures of drilling a mold (FIG. 7A), casting,e.g., silicon rubber (FIG. 7B) to prepare a silicon negative (FIG. 7C),placing the negative in a mold and and casting the polymer solution(FIG. 7D), thousands of nano-wells (submicroliter volume) can beproduced in a small gel plate (FIG. 7E) for handling of submicrolitersample volumes. In use, a nano-well gel plate, such as that of FIG. 7E,is partially immersed in a buffer solution. In spite of the extremelysmall volumes deposited, evaporation is compensated for by watersupplied by liquid flow into and through the surrounding gel matrix.

[0035] The angle of capillary exit end orientation in relation to theopening of individual wells is an important parameter for ease of samplecollection. Orientation at an angle, as shown in FIG. 8A, allowscontinuous electrical contact to be maintained with the gel surfacesurrounding traditionally shaped wells. (Refer also to FIG. 9A.) Acapillary with a vertically oriented tip, as shown in FIG. 8B, can bepositioned more precisely and can be moved in any direction; however,when a vertically oriented capillary tip is moved from one traditionallyshaped well to another, the electrical contact with the surface of thegel is often broken. To address this problem, we have designed “nozzle”shaped wells, as shown in FIG. 9B and FIGS. 10A-10D. In thisconfiguration, the outer top edges of the individual wells areparticularly close together; a vertically oriented capillary tip cansimply be pushed through the fluid gel from one well to another, underthe surface of the buffer, maintaining the electrical connection.

[0036] Since the properties of the gel can easily be modified bychanging gel concentration, crosslinking or chemically modifiying thegel, functions difficult to incorporate with standard materials may bepossible. For example, electrophoretic separation can be performed in agel with an array of wells and the separated substances can easily beremoved from the wells without tedious extraction from the gel. Poreand/or pH gradient gels would be especially beneficial for thisapplication, e.g., for protein preparation. For example, Immobilin™ canbe used as a gel matrix for micropreparative isoelectric focusing. Ofcourse, other functionalized gels may be used. For example, immobilizedantibody or antigen containing gels may be used for affinity capture.Since channels and wells of practically any shape can be easilyfabricated by gel casting, many structures fabricated in “classicalchips” can be fabricated in gel more cost effectively. In addition,enzymes can be immobilized in the gel structure, and reactions such asdigestion (protein or DNA), PCR and sequencing can be carried out. Thegel can be heated, e.g., by microwaves, if necessary. If the enzymes(substrates, template, . . .) are immobilized in the gel, little or nosample cleanup would be necessary compared to other sample handlingsystems. ssDNA can be fixed in the gel for specific hybridization to acomplementary DNA strand. In addition, other biospecific groups such asantibodies could also be immobilized in individual wells. In particular,inert particles, such as beads, can be placed in individual wells, ascarriers of active materials, e.g., antibldies, enzymes, substrates,etc.. For, example, functionalized solid phase particles would be usefulfor on-plate combinatorial chemical analysis.

[0037] Besides bare gel blocks casted or molded for the purposesdescribed above, other contemplated uses for hydrophilic gels are ascomponents of “cassettes.” Such cassettes could be hybrid gel-plastic orgel-glass or gel-metal devices or chips, where a mold serves as a gelplate enclosure. All surfaces of the gel block would be covered, besideschannels and wells. This design would both prevent excessive losses ofwater from the gel during sample manipulation (e.g., microwave heating)and ease the handling of the gel devices. A mold would be made as areusable device, which would significantly reduce costs, especially ifthe mold contains embedded contacts or heating elements.

[0038] The following example is presented to illustrate the advantagesof the present invention and to assist one of ordinary skill in makingand using the same. These examples are not intended in any way otherwiseto limit the scope of the disclosure.

EXAMPLE

[0039] To verify that individual components of a sample can be collectedusing the system of the invention with a microtiter multi-wellcollection plate, an experiment was conducted with a fluorescentlylabelled double stranded DNA restriction fragment mixture (commerciallyavailable as pBR322/Hinf I) as a sample. The mixture was separated bycapillary electrophoresis (CE) in a 75 im i.d. polyvinylalcohol coatedfused silica capillary filled with linear polyacrylamide (4% solution in50 mM Tris/TAPS buffer) in an electric field of 370V/cm. The totalcapillary length was 27 cm and the length from injection to detectionpoint was 25 cm. Injection was performed electrokinetically for 2-3seconds at 370 V/cm. Detection was accomplished on-column by laserinduced fluorescence using an argon ion laser (488 nm) and emission at520 nm by means of confocal detection. The microtiter gel collectionplate was a 3% agarose composite (a mixture of 1.5% large pore and 1.5%narrow pore agarose material), containing a single lane of microwells.During separation, the capillary was moved from one microwell to anotherin constant time intervals of 30 seconds. After deposition, thefractions were transferred out of the microwells, desalted andidentified by re-injection and capillary electrophoresis. FIG. 11 showsthe detector signal during the original separation analysis of thecollected fractions. The 30 second collection time intervals aredepicted as vertical lines, with the fractions labelled from 1 to 15.FIG. 12 shows the results after re-injection of all the collectedfractions (fraction 1 through 8 in FIG. 12A and fractions 9 through 16in FIG. 12B). The results clearly show individually collected fractionswith no contamination between fractions. The final profile depicted atthe end of each of FIGS. 12A and 12B shows that the pooled fractionscontain all of the components of the individual fractions. FIG. 13 showsanother successful fraction collection experiment from a differentsample.

[0040] While the present invention has been described in conjunctionwith a preferred embodiment, one of ordinary skill, after reading theforegoing specification, will be able to effect various changes,substitutions of equivalents, and other alterations to the compositionsand methods set forth herein. It is therefore intended that theprotection granted by Letters Patent hereon be limited only by thedefinitions contained in the appended claims and equivalents thereof.

What is claimed is:
 1. A solvent permeated gel for a microfabricated device comprising an entangled or crosslinked polymeric network; and an excess of a solvent permeating said polymeric network, wherein said solvent permeated gel has sufficient dimensional rigidity and stability to maintain a specific shaped compartment microfabricated into said gel and wherein said excess of solvent permeating said polymeric network is sufficient to reduce or eliminate evaporation of liquid from any said compartment.
 2. The solvent permeated gel of claim 1, wherein said polymeric network comprises agarose or polyacrylamide.
 3. The solvent permeated gel of claim 1, wherein said polymeric network is a composite of polymers having different pore sizes.
 4. The solvent permeated gel of claim 1, wherein said solvent comprises water.
 5. The solvent permeated gel of claim 1, wherein said solvent comprises an organic solvent or a mixed aqueous/organic solvent.
 6. A microfabricated device comprising a solvent permeated gel, said gel comprising an entangled or crosslinked polymeric network and an excess of a solvent permeating said polymeric network, wherein a plurality of microscale compartments are fabricated in said solvent permeated gel, said gel further has sufficient dimensional rigidity and stability to maintain the shape of said compartments and said excess of solvent permeating said polymeric network is sufficient to reduce or eliminate evaporation of liquid from said compartments.
 7. The microfabricated device of claim 6, wherein said solvent permeated gel comprises immobilized buffer components and said device is configured for micropreparative isoelectric focusing.
 8. The microfabricated device of claim 6, wherein a pH gradient is established in said gel across said device.
 9. The micro fabricated device of claim 6, wherein individual said compartments comprise an antibody or an antigen immobilized in said compartment.
 10. The microfabricated device of claim 6, wherein individual said compartments comprise an enzyme immobilized in said compartment.
 11. The microfabricated device of claim 6, wherein individual said compartments comprise nucleic acid immobilized in said compartment.
 12. The microfabricated device of claim 6, wherein individual said compartments comprise inert particles to which active elements can be attached immobilized in said compartment.
 13. The microfabricated device of claim 6, wherein said particles are magnetic beads.
 14. The microfabricated device of claim 6, wherein individual said compartments in said device are configured as channels.
 15. The microfabricated device of claim 6, wherein individual said microscale compartments in said device are configured as wells.
 16. The microfabricated device of claim 6, wherein individual said microscale compartments in said device are configured as nozzle-shaped wells.
 17. The microfabricated device of claim 6, wherein said polymeric network comprises agarose or polyacrylamide.
 18. The microfabricated device of claim 6, wherein said polymeric network is a composite of polymers having different pore sizes.
 19. The microfabricated device of claim 6, wherein said solvent comprises water.
 20. The microfabricated device of claim 6, wherein said solvent comprises an organic solvent or a mixed aqueous/organic solvent.
 21. A microfabricated device comprising a solvent permeated gel, said gel consisting essentially of an entangled or crosslinked polymeric network and an excess of a solvent permeating said polymeric network, wherein a plurality of microscale compartments are fabricated in said solvent permeated gel, said gel further has sufficient dimensional rigidity and stability to maintain the shape of said compartments and said excess of solvent permeating said polymeric network is sufficient to reduce or eliminate evaporation of liquid from said compartments. 